U.S. patent application number 11/283365 was filed with the patent office on 2007-05-24 for image based correction for unwanted light signals in a specific region of interest.
Invention is credited to Gavin A. Cloherty, Robert C. Gray, Shihai Huang, James C. Kolterman, Eric B. Shain.
Application Number | 20070116376 11/283365 |
Document ID | / |
Family ID | 37770289 |
Filed Date | 2007-05-24 |
United States Patent
Application |
20070116376 |
Kind Code |
A1 |
Kolterman; James C. ; et
al. |
May 24, 2007 |
Image based correction for unwanted light signals in a specific
region of interest
Abstract
A method for correcting the signal in an image having a
plurality of regions of interest, the method comprising the steps
of: (a) providing an image having a plurality of regions of
interest, these regions of interest having areas between them; (b)
determining a region of correction between at least two regions of
interest; (c) calculating a correction signal from the region of
correction; and (d) using the correction signal to correct a
measured signal from one or more regions of interest. This
invention also provides a method for defining a region of
correction for use in a method for correcting the signal in an
image having a plurality of regions of interest, the defining
method comprising the steps of: (a) providing an image having a
plurality of regions of interest; (b) extracting geometric
information for a plurality of regions of interest; (c) selecting a
location between at least two regions of interest; (d) selecting at
least one parameter to describe regions of correction; and (e)
constructing regions of correction between the at least two regions
of interest.
Inventors: |
Kolterman; James C.;
(Libertyville, IL) ; Shain; Eric B.; (Glencoe,
IL) ; Gray; Robert C.; (Gurnee, IL) ; Huang;
Shihai; (Evanston, IL) ; Cloherty; Gavin A.;
(Wauconda, IL) |
Correspondence
Address: |
ROBERT DEBERARDINE;ABBOTT LABORATORIES
100 ABBOTT PARK ROAD
DEPT. 377/AP6A
ABBOTT PARK
IL
60064-6008
US
|
Family ID: |
37770289 |
Appl. No.: |
11/283365 |
Filed: |
November 18, 2005 |
Current U.S.
Class: |
382/274 ;
382/128 |
Current CPC
Class: |
G06T 7/11 20170101; G06T
2207/30072 20130101; G16B 25/00 20190201 |
Class at
Publication: |
382/274 ;
382/128 |
International
Class: |
G06K 9/40 20060101
G06K009/40; G06K 9/00 20060101 G06K009/00 |
Claims
1. A method for correcting the signal in an image having a
plurality of regions of interest, the method comprising the steps
of: (a) providing an image having a plurality of regions of
interest, these regions of interest having areas between them; (b)
determining a region of correction between at least two regions of
interest; (c) calculating a correction signal from the region of
correction; and (d) using the correction signal to correct a signal
measurement from one or more regions of interest.
2. The method of claim 1, further including the step of determining
a background signal and adjusting the correction signal of a run by
subtracting the background signal from the correction signal.
3. The method of claim 2, wherein the background signal is a
background signal stored prior to commencing the run.
4. The method of claim 2, wherein the background signal is a
background signal determined during the run.
5. The method of claim 1, wherein the correction signal is
scaled.
6. The method of claim 1, wherein the regions of correction have a
plurality of sides.
7. The method of claim 1, wherein the regions of correction have
four sides.
8. The method of claim 1, wherein the regions of correction are
closed polygons.
9. The method of claim 1, wherein the regions of correction are
circular.
10. The method of claim 1, wherein the regions of correction are
annular.
11. The method of claim 1, wherein the regions of correction are
defined by a bitmap.
12. The method of claim 1, wherein said plurality of regions of
interest are from a multi-well plate.
13. The method of claim 1, wherein a thermocycler reader is
employed.
14. The method of claim 1, further including the step of storing
the regions of correction defined in step (c).
15. A method for defining a region of correction for use in a
method for correcting the signal in an image having a plurality of
regions of interest, the defining method comprising the steps of:
(a) providing an image having a plurality of regions of interest;
(b) extracting geometric information for the plurality of regions
of interest; (c) selecting a location between at least two regions
of interest; (d) selecting at least one parameter to describe
regions of correction; and (e) constructing regions of correction
between the at least two regions of interest.
16. The method of claim 15, further including the step of
constructing additional regions of correction that are not between
the at least two regions of interest.
17. The method of claim 16, wherein a sufficient number of
additional regions of correction are constructed so that each
region of interest has the same number of regions of correction as
does any other region of interest.
18. The method of claim 15, wherein the geometric information for
the regions of interest is a centroid.
19. The method of claim 18, wherein the location between at least
two regions of interest is the center point between the centroids
of at least two regions of interest.
20. The method of claim 15, wherein the regions of correction are
selected from the group consisting of polygons, circles, annuli,
and bitmaps.
21. The method of claim 15, further including the step of storing
the regions of correction associated with the at least two regions
of interest.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to a method for correcting signals
detected by a detection system in a diagnostic instrument.
[0003] 2. Discussion of the Art
[0004] Raw images generated by a diagnostic instrument having a
digital image sensor as a detector, such as, for example, the
Applied Biosystems Prism 7000 diagnostic instrument, can exhibit an
anomaly known as "cross-talk." Cross-talk refers to the situation
where a signal from a given location in the image (for example, a
given well in a plate having a plurality of wells, e.g., a 96-well
PCR plate), causes a variation in the signal at a different
location in the image (for example, a different well in the plate
having a plurality of wells). A specific region within an image
associated with an independent signal is often referred to as a
region of interest (alternatively referred to herein as ROI). Each
ROI defines the specific pixels within the image associated with a
specific reaction. Variations in signal due to cross-talk, although
typically small, can induce variations in reaction quantification
of one or more regions of interest within the image. In some cases,
sensitivity of the reaction is reduced by requiring an increase in
signal threshold in order to avoid false positive results due to
cross-talk.
[0005] The areas in the image between the regions of interest of
the image contain optical information that can be used to
compensate for sources of variation in signal. These sources of
signal variation can result from a specific geometric optical
reflection, scattered light from optical components, light leakage,
changes in intensity of the source of light, and the like. All of
these sources of variation can contribute to a dynamically changing
error in the optical signal in a given region of interest of the
image.
[0006] It is desired to monitor a region of interest associated
with a reaction and ultimately correct for varying anomalous
signals over the course of a testing run in a diagnostic
instrument.
SUMMARY OF THE INVENTION
[0007] In one aspect, this invention provides a method for
correcting the signal in an image having a plurality of regions of
interest, the method comprising the steps of: [0008] (a) providing
an image having a plurality of regions of interest, these regions
of interest having areas between them; [0009] (b) determining a
region of correction between at least two regions of interest;
[0010] (c) calculating a correction signal from the region of
correction; and [0011] (d) using the correction signal to correct a
measured signal from one or more regions of interest.
[0012] In another aspect, this invention provides a method for
defining a region of correction for use in a method for correcting
the signal in an image having a plurality of regions of interest,
the defining method comprising the steps of: [0013] (a) providing
an image having a plurality of regions of interest; [0014] (b)
extracting geometric information for a plurality of regions of
interest; [0015] (c) selecting a location between at least two
regions of interest; [0016] (d) selecting at least one parameter to
describe regions of correction; and [0017] (e) constructing regions
of correction between the at least two regions of interest. The
regions of correction defined in the forgoing method can be stored
for further use to correct signals measured from one or more
regions of interest.
[0018] The specified regions of correction can have various shapes,
such as, for example, circles, squares, diamonds, rectangles, or
other geometric figures. Storing of the regions of correction
involves determining the definition of the location and the shape
of the geometric areas and specifying the pixels contained within
each area.
[0019] The method of this invention can be used to measure a
dynamically changing signal and the effect of the dynamically
changing signal on a region of interest of a specific reaction.
Correcting for the cross-talk inherent in a dynamically changing
signal will greatly increase the sensitivity of the method of
detection used in an assay employing such signals. The method of
this invention does not affect the optical path of the light
collected by a detector. The method can be applied directly to an
image that is collected for all the regions of interest
[0020] By measuring the signals in the regions of correction, the
signal anomaly due to cross-talk can be significantly reduced.
[0021] The sizes and shapes of the regions of correction can vary
and depend primarily on the orientation of the existing regions of
interest and any image distortions that may be present.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a flow chart that defines the placement of
diamond-shaped regions of correction about circular reaction
regions of interest.
[0023] FIG. 2A illustrates one embodiment of reaction regions of
interest and regions of correction. The reaction regions of
interest are circular and the regions of correction are circular.
FIG. 2B illustrates another embodiment of reaction regions of
interest and regions of correction. The reaction regions of
interest are circular and the correction regions of interest are
rectangular. FIG. 2C illustrates still another embodiment of
reaction regions of interest and regions of correction. The
reaction regions of interest are circular and the regions of
correction are shaped like diamonds.
[0024] FIG. 3 is a flow chart illustrating the application of the
image-based correction algorithm of this invention.
[0025] FIG. 4 is a map of a 96-well plate illustrating the location
of each positive response and each negative response.
[0026] FIG. 5 is a sample of an image at the end of a run.
[0027] FIG. 6 is a sample of an image showing reaction regions of
interest and regions of correction.
[0028] FIG. 7 shows fluorescence responses in a PCR assay without
image-based correction applied.
[0029] FIG. 8 shows fluorescence responses in a PCR assay without
image-based correction applied. In this figure, the Y scale is
expanded.
[0030] FIG. 9 shows fluorescence responses in a PCR assay with
image-based correction. In this figure, the Y scale is
expanded.
[0031] FIG. 10 shows the fluorescence response in a PCR assay for
well F-11 from FIG. 4 with and without assay-based correction.
DETAILED DESCRIPTION
[0032] As used herein, the expression "region of interest" means
the collection of pixels within an image that define the location
of a specific optical signal. The expression "reaction region of
interest" means the region of interest associated with a specific
reaction in an assay. The expressions "region of correction" and
"correction region of interest" mean the area associated with the
background portion of the image adjacent to a reaction region of
interest. The expression "reaction pixel sum" means the sum of all
the pixel intensity values within a reaction region of interest.
The expression "reaction pixel count" means the number of pixels
within a reaction region of interest. The expression "reaction
region of interest pixel average" means the value obtained by
dividing the reaction pixel sum by the reaction pixel count. The
expression "correction pixel sum" means the sum of all the pixel
intensity values within a region of correction. The expression
"correction pixel count" means the number of pixels with a region
of correction. The expression "region of correction pixel average"
means the value obtained by dividing the correction pixel sum by
the correction pixel count. The term "scale" means a multiplicative
factor applied to the correction calculation. The term "centroid"
means the geometric center of a region of interest. As used herein,
the terms "circular", "rectangular", "annular", and other terms
relating to shape referred to herein are intended to include shapes
that are substantially circular, substantially rectangular,
substantially annular, and shapes that are substantially similar to
the other shapes referred to herein, respectively.
[0033] In one aspect, this invention provides a method for
correcting an image having a plurality of reaction regions of
interest and a plurality of regions of correction, the method
involving the steps of: [0034] (a) providing an image having a
plurality of regions of interest, these regions of interest having
areas between them; [0035] (b) determining a region of correction
between at least two regions of interest; [0036] (c) calculating a
correction signal from the region of correction; and [0037] (d)
using the correction signal to correct a signal measurement from
one or more regions of interest. Prior to carrying out the method
of this invention, certain steps must be taken to calibrate the
imaging system, which is typically a digital imaging system. FIG. 3
shows a flow chart that illustrates steps for defining regions of
correction between adjacent reaction regions of interest for the
calibration step of the method of this invention. In this flow
chart, generic regions of correction are described.
[0038] According to the calibration method of this invention, the
centroid of each reaction region of interest is determined. The
reaction regions of interest are typically determined by using a
calibration where signals in a device having a plurality of
reaction sites are measured. A signal is measured at each reaction
site. In the case of 96-well reaction plates, the signals in a
calibration plate containing fluorescent dye at each reaction site
can be measured by an imaging sensor. A calibration plate is a
96-well reaction plate used for calibrating the instrument used.
The reaction regions of interest can be determined by locating the
contiguous pixels at each reaction site within the image. The
geometric centroid of each set of centroids from four adjacent
reaction regions of interest can be used to determine a center
point for a region of correction. A region of correction using that
center point and a specific geometric shape can be defined. As
shown in FIGS. 2A, 2B, and 2C, the reaction regions of interest are
circular in shape. A region of correction can be circular-shaped,
as shown in FIG. 2A, rectangular-shaped, as shown in FIG. 2B, or
diamond-shaped, as shown in FIG. 2C. Other shapes, such as, for
example, closed polygons, are suitable for both the reaction
regions of interest and the regions of correction. The parameters
of the regions of correction are typically radii of rings for
circular-shaped regions of correction, length and width for
rectangular-shaped regions of correction, and length of sides for
diamond-shaped regions of correction. Dimensions for the particular
geometric shape selected are specified. An alternative to defining
regions of correction by means of geometric shapes involves the use
of an arbitrary bitmap. Such a bitmap could, for example, be a 9 by
9 array of values specifying which pixels would be included in the
region of correction and which pixels would be excluded from the
region of correction. The center points of the regions of
correction can be mirrored to create regions of correction on the
periphery of the plate for the outer rows and columns of the
reaction regions of interest in the image. Thus, in the case of
diamond-shaped regions of correction in an image having 96 reaction
regions of interest, there are 117 diamond-shaped regions of
correction of interest in total, i.e., four (4) diamond-shaped
regions of correction per reaction region of interest. The use of
diamond-shaped regions of correction is shown in FIG. 2C. The
regions of interest associated with specific wells can be
determined and stored, such as, for example, by means of a
computer. In this embodiment, each reaction region of interest has
the four adjacent regions of correction associated with it.
[0039] Similarly, in the case of rectangular-shaped regions of
correction in an image having a plurality of reaction regions of
interest (e.g., 96 wells in a plate), the rectangles can be
oriented with the length parallel to the x-axis or to the y-axis,
as shown in FIG. 2B. For the x-direction (horizontal), the center
point between two adjacent regions of interest is located. A
rectangle is constructed by using the center point between two
adjacent regions of interest as the center of the region of
correction between the regions of interest. For the y-direction
(vertical), the center point between two adjacent regions of
interest is located. The rectangle is constructed by using the
center point between two adjacent regions of interest as the center
of the region of correction between the regions of interest.
Rectangles are also created on the periphery of the image for the
outer rows of regions of interest and outer columns of regions of
interest. The mirror of the center between adjacent regions of
interest is used to set the center of the region of correction
rectangle. The regions of interest associated with specific wells
can be determined and stored. In this embodiment, each reaction
region of interest has the four adjacent regions of correction
associated with it. Measures other than the centroid of the regions
of correction can also be used to define the location of regions of
correction. For example, the region of correction can be placed
equidistant from boundaries of adjacent regions of interest.
[0040] After the region of correction calibration is performed, the
correction based upon from the region of correction can be applied
by using the following method. Once the region of correction
calibration is performed, a background offset value needs to be
generated. This value can be generated in at least two ways.
According to a first alternative, a background calibration can be
performed. In this method, an image is taken of a plate without any
fluorescent dye. During the background calibration, the average
pixel value for each region of correction is calculated by dividing
the pixel sum by the pixel count in that region of correction to
obtain an average pixel value. This average pixel value is
indicative of the background light level and is referred to as the
background offset value. The background offset values are stored
for use in future runs, e.g., PCR runs. Alternatively, the
background offset value can be determined on a run-by-run basis by
calculating the average pixel value for each region of correction
for the first reading of a run, e.g., a PCR run. Because the first
(or first few) readings of a PCR run occur before a significant
reaction signal is produced, this alternative method provides a
good representation of the background.
[0041] The signal correction is performed in the following manner.
Performance of signal correction is depicted in FIGS. 1 and 3. For
each reaction, the reaction pixel sum and the reaction pixel count
are calculated by using the reaction region of interest. The
average pixel value for the four regions of correction associated
with a given reaction region of interest is calculated. Although
four regions of correction are shown in FIG. 1 and diamond-shaped
regions of correction are shown in FIG. 1, the method is not
limited to four regions of correction nor is the invention limited
to diamond-shaped regions of correction. The background offset
value is subtracted from the region of correction pixel average.
Then, this difference is multiplied by the reaction region of
interest pixel count and, if necessary, by a scale factor, to
generate a correction value. The correction value is then
subtracted from the reaction region of interest pixel sum to
generate a corrected reaction region of interest pixel sum. The
scale factor is typically dependent upon the detection system. An
example of a scale factor is 1.15. In some instrument systems,
multiple exposures are made at each reading to increase the dynamic
range of measurement. In this case, a corresponding background
offset and region of correction pixel average needs to be generated
for each exposure. The correction to the reaction pixel sum is then
made for the exposure of longest duration that does not exhibit
significant saturation of the image sensor.
[0042] This invention can also be applied to an assay system based
on array or a microarray, such as, for example, the Vysis
GenoSensor genomic DNA microarray system (Abbott Laboratories,
Abbott Park, Ill.). Such systems can measure a plurality of genomic
targets through hybridization to an array of capture targets placed
on a surface, such as, for example, a glass "chip" or a microscope
slide. The product of the hybridization is typically measured by
means of fluorescent dyes and an electronic imaging system.
[0043] The following non-limiting example further explains the
method of this invention.
EXAMPLE
[0044] A real time PCR run for HIV was performed on an ABI Prism
7500 instrument (Applied Biosystems, Foster City, Calif.). This
instrument utilizes a 96-well plate format with wells arranged in a
12.times.8 array. The run was configured so that there were 84
wells containing positive samples with a concentration of
1.times.10.sup.6 copies/mL and 12 wells not containing positive
samples, i.e., negative wells. The negative wells were distributed
on the plate to maximize the potential cross-talk from the wells
containing positive samples. FIG. 4 illustrates the layout of the
plate.
[0045] The ABI Prism 7500 instrument uses a CCD camera and measures
fluorescence in five wavelength bands. FIG. 5 shows one image from
the end of the PCR run. FIG. 6 shows the same image with the
reaction regions of interest and the regions of correction
superimposed. In this example, a diagonal array of diamond-shaped
regions of correction, each of which contained of 25 pixels, were
used. The first reading in the PCR run was used to establish the
background offset values for each subsequent reading. The scaling
factor used was 1.15.
[0046] FIG. 7 shows the raw fluorescence signals for all 96 samples
without any image-based correction applied. As can be seen, the 84
positive samples generated signals significantly above the
background fluorescence by cycle 15 and approached their maximum
fluorescence by cycle 25. FIG. 8 shows the same responses with the
Y-axis scaled to focus on the responses in wells not containing
positive samples. All of the negative responses showed a small but
significant rise from cycles 15 through 25, which is caused by
cross-talk from the responses of the positive samples. FIG. 9 shows
the effect of the image-based correction on the negative responses.
As can be seen, the cross-talk signal has been effectively
eliminated. FIG. 10 shows the response for well F-11 with and
without the image-based correction applied.
[0047] The method is also applicable to images that contain a fewer
or a greater number of regions of interest.
[0048] Various modifications and alterations of this invention will
become apparent to those skilled in the art without departing from
the scope and spirit of this invention, and it should be understood
that this invention is not to be unduly limited to the illustrative
embodiments set forth herein.
* * * * *